The long-term objective of this work is to understand how a eukaryotic chromosome replicates. To this end, a replication map of chromosome III of the yeast, Saccharomyces cerevisiae, has been prepared. This map includes the positions of ARS elements, detected by their ability to promote the autonomous replication of plasmids, the positions of DNA replication origins, detected by 2D gel analysis of replication intermediates, and the positions of DNA replication termination. This proposal addresses three major issues concerning the replication and maintenance of chromosome III. First, both genetic and molecular approaches will be used to study the mechanism(s) by which a derivative of this chromosome lacking active chromosomal replicators. Second, the mechanism(s) by which chromosomal replicator activity is influenced by chromosomal context will be studied by examining the temporal pattern of replication of the chromosome and by using several genetic and molecular approaches designed to map the determinants of replicator activity. Finally, the structure of several chromosome III replicators will be further defined using both genetic and biochemical approaches. The binding sites for the replicator initiator protein, ORC, will be identified, and novel proteins that bind to replicators will be sought with a genetic screen. Replication timing determinants in ARS301 will be identified, and the role of ARS304 in the function of the recombination enhancer that activates HML as a donor of information in mating type switching will be analyzed. These studies will provide new insights into a fundamental cellular process, chromosome replication. Knowledge gained from the genetically tractable yeast system is applicable to larger eukaryotes, and guides the approaches to these experimentally difficult systems. The proposed studies are also likely to lead to a further understanding of chromosome rearrangements and the reactivation of DNA replication that are characteristics of malignant cells.